Mass spectrometry (MS)-based absolute targeted protein quantification is being increasingly adopted in proteomics research, and has proven its utility in both basic research and clinical biomarker discovery and validation. Several approaches have been developed to determine absolute quantity of targeted proteins in various biological samples based on the use of different stable isotype-labeled internal standards (IS), such as synthetic peptides (AQUA) (Gerber et al., Proc Natl Acad Sci USA 2003, 100, (12), 6940-5; Keshishian et al., Mol Cell Proteomics 2009, 8, (10), 2339-49) quantification concatemers (QconCATs) (Beynon et al., Nat Methods 2005, 2, (8), 587-9; Pratt et al., Nat Protoc 2006, 1, (2), 1029-43) and full-length protein standards (PSAQ) (Hanke et al., J Proteome Res 2008, 7, (3), 1118-30; Brun et al., Mol Cell Proteomics 2007, 6, (12), 2139-49). A combination of these approaches with modern MS/MS technologies, mainly multiple reaction monitoring (MRM), permits high accuracy and precision in absolute protein quantification.
To perform AQUA, the most commonly used absolute protein quantification method, isotopically labeled synthetic peptides are added to digested protein samples as the IS, followed by peptide extraction and MS analysis. Protein quantity is determined according to the peak ratios of unlabeled natural peptides to their heavy labeled counterparts. AQUA is based on the assumptions that 1) the targeted proteins are fully recovered after extraction; 2) the surrogate peptides are fully digested from targeted proteins in samples and remain intact during enzymatic digestion. However, these assumptions have never been fully verified for most proteins/peptides. Additionally, synthesis and purification of isotope labelling peptides can be expensive and time consuming.
A recent development of absolute protein quantification methods is the QconCATs (Beynon et al., supra; Pratt et al., supra). QconCATs are artificial proteins made of a number of concatenated surrogate peptides for various analyte proteins. Isotope-labeled QconCATs are obtained via the expression of artificial QconCATs genes in cells cultured in a heavy isotope enriched medium. QconCATs are added to protein samples before digestion, and the digested concatenated peptides serve as IS for quantification of different proteins. A major pitfall of this approach is that protein extraction and digestion efficiencies could vary significantly between QconCATs and native proteins due to different amino acid compositions.
Protein standards for absolute quantification (PSAQ) is an alternative approach of the AQUA and QconCATs. PSAQ involves biosynthesis and purification of stable isotope labeled analyte proteins and the addition of a known quantity of labeled proteins to samples (Hanke et al, supra; Brun et al., supra). PSAQ can overcome some drawbacks of AQUA, such as the variations associated with protein extraction and digestion. However, PSAQ is generally a low throughput assay, and each PSAQ protein standards has to be generated and purified individually, which is very labor intensive and expensive.
In addition to the stable isotope labelling-based methods, so-called “label-free” absolute quantification methods have been developed using unlabeled proteins as the external standard (Mayr et al., J Proteome Res 2006, 5, (2), 414-21; Arike et al., J Proteomics 2012, 75, (17), 5437-48). However, this approach is generally considered unfavorable in terms of accuracy and precision when compared with the methods employing isotopically labeled IS.
Improved methods of protein quantitation are needed.